The review of catalyst applications was supported by the U.S. Department of Energy, Office of Science, Office of Basic Energy Sciences, Division of Chemical Sciences, Biosciences, and Geosciences Catalysis Program under contract DE-AC05-RL01830 and FWP-47319. The review of biomedical applications was supported by the National Institute of Health, National Institute of Environmental Health Sciences under grant R21ES029778. Experiments were conducted at EMSL (grid.436923.9), a DOE Office of Science User Facility sponsored by the Office of Biological and Environmental Research and located at Pacific Northwest National Laboratory (PNNL). PNNL is a multi-program national laboratory operated by Battelle for the U.S. Department of Energy under contract DE-AC05-RL01830 and FWP-47319.

The protocol is divided into four sections which specify 1) the preparation of any solid materials being used in the system or activation or clearing of undesired adsorbed species, 2) addition of the solid and liquid materials to the NMR rotor, 3) addition of gases to the rotor, and 4) conducting the NMR experiments in the spectrometer. The procedure is representative of a typical sequence but may be modified to fit the specific needs of the experiment.
1. Pretreating solid samples
<ol>
	<li>Weigh approximately twice the mass of the solid sample that is desired for the NMR experiment (for a 7.5 mm rotor, ~250 mg) and place the solid sample into a quartz sample tube used for treating materials in a furnace system, plugging the tube with quartz wool to hold the material in place.</li>
	<li>Connect the isolation valves to the solids treatment 1) flow or 2) vacuum system by placing the tube into the cool furnace and tightening the connections.</li>
	<li>Affix the quartz tube end(s) onto the gas isolation valve(s) in the open position.</li>
	<li>Begin the treatment.
	<ol>
		<li>For flow systems:
		<ol>
			<li>Affix a thermocouple to the outside of the tube, holding it in place with a heat-resistant material.</li>
			<li>Begin the flow of the treatment gas (e.g., N2 at 100 sccm) to clear the solid surface or activate the material.</li>
		</ol>
		</li>
		<li>Alternatively, for vacuum systems:
		<ol>
			<li>Close the isolation valve to the vacuum system and start the vacuum pump.</li>
			<li>When full vacuum is established, very slowly open the isolation valve to apply vacuum to the sample, pausing periodically to allow the system to equilibrate. Continue until the valve is open.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Turn on the furnace controller and set the temperature ramp program to the desired condition (e.g., 300 &#176;C for 4 hours at a ramp rate of 5 &#176;C/min).</li>
	<li>Start the temperature program and let it run.</li>
	<li>When completed, allow the sample to cool to a workable temperature.</li>
	<li>Turn off the temperature controller and stop the flow/vacuum.</li>
	<li>Quickly seal the sample with the isolation valves to maintain the desired sample environment.</li>
	<li>Disconnect the quartz tube from the treatment system and transfer the tubes and closed valves to the antechamber of a dry, N2-purged glove box.</li>
	<li>Empty and refill the antechamber at least 4 times and transfer the tube inside of the glove box.</li>
</ol>
2. Loading solid samples into the NMR rotor
<ol>
	<li>Weigh the empty and clean high-pressure, high-temperature NMR rotor with the rotor cap.</li>
	<li>Place the NMR rotor in the holder to maintain directionality.</li>
	<li>Place the sample funnel into the bore of the rotor.</li>
	<li>Remove the isolation valve(s) from the sample tube and pour a small quantity of solid material into the funnel.</li>
	<li>Tap the powder down into the funnel and lightly direct it into the rotor with the packing rod as necessary.</li>
	<li>Repeat the stepwise addition of solid material until the desired quantity (e.g., &#189; rotor) is achieved.</li>
	<li>Weigh the NMR rotor (and cap) with the sample inside to determine the quantity of sample added.</li>
	<li>If desired, draw up a specified quantity of any liquid sample and slowly inject the liquid into the center of the NMR rotor with a micro syringe.</li>
	<li>Seal the rotor by placing the cap onto the top and turning it counterclockwise with the rotor cap bit to engage the O-ring between the rotor and cap. Note that a new O-ring may be&#160;periodically required to prevent leaking, especially if using chemically abrasive mixtures or small gases such as hydrogen.</li>
	<li>Weigh the NMR rotor to determine the total mass of added sample.</li>
</ol>
3. Charging the NMR rotor with the desired chemicals at the desired conditions
<ol>
	<li>Place the sealed NMR rotor into the rotor stage, ensuring the size of the stage insert is compatible with the rotor size, and tighten the nut by hand to secure it in place. Note that the tightness of the rotor in the holder in this step will determine the tightness of the cap seal.</li>
	<li>Lower the rotor stage into the lower section of the high-pressure exposure device.</li>
	<li>Use an Allen wrench to turn one of the screws 90&#176; to secure the rotor stage into the bottom of the exposure device.</li>
	<li>Place the top section of the NMR loading device into and on top of the bottom section, lining up the NMR cap bit to the top of the cap head of the NMR rotor to ensure it is engaged.</li>
	<li>Place the 2 clamps over the top of the lip where the upper and lower sections of the exposure device meet and latch them in place.</li>
	<li>Tighten the 6 bolts on the top of the upper section of the exposure device to engage the sealing surface between the upper and lower sections.</li>
	<li>Connect the upper section of the NMR exposure device to the gas line inlet and outlets.</li>
	<li>Connect the thermocouple on the upper section of the NMR exposure device to the temperature sensor.</li>
	<li>If desired, wrap the heating tape around the gas lines and upper sections of the exposure device to enable heating with the respective controller. A hot plate can also be engaged.</li>
	<li>Ensuring the exposure chamber outlet is open and source gas valve is closed, turn on the vacuum pump to remove air from the exposure device and associated lines.</li>
	<li>Purge the lines with either the desired gas or an inert one, cycling between vacuum and atmospheric pressure three times to ensure the lines are cleared of air.</li>
	<li>Prepare the desired gas composition either from 1) a high-pressure delivery system or 2) a flow system to introduce vapors at a specified pressure.
	<ol>
		<li>For high-pressure or vacuum sample preparation:
		<ol>
			<li>Close the exposure device gas outlet and set the gas manifold valves to bypass the liquid injection line.</li>
			<li>Set the desired pressure on the high-pressure syringe pump of the high-pressure delivery system.</li>
			<li>Open the gas source valves on the high-pressure syringe pump and run the program set on the pump, monitoring the real pressure inside of the exposure device.</li>
			<li>When the desired pressure is achieved inside the exposure device, stop the syringe pump and close the source gas valves.</li>
			<li>Open the NMR rotor by rotating clockwise the external screw mechanism, which is coupled to the interior NMR cap bit.</li>
			<li>Allow the gas of the desired pressure to enter the NMR rotor and equilibrate.</li>
			<li>Reseal the NMR rotor by rotating the external screw mechanism counterclockwise. A viewing window will assist in determining when the rotor is closed.</li>
			<li>Slowly depressurize the system by opening the exposure device gas outlet valve.</li>
		</ol>
		</li>
		<li>For flowing gas or vapor sample preparation:
		<ol>
			<li>Ensure the exposure device gas outlet is open to prevent over-pressure.</li>
			<li>Set the desired gas flow rate on the mass flow controller and begin the gas flow.</li>
			<li>Connect the liquid supply line from the liquid syringe pump to the gas manifold.</li>
			<li>Set the gas manifold valves to enable flow to the liquid injection line.</li>
			<li>Set the liquid flow rate on the liquid syringe pump to achieve the desired vapor pressure and begin liquid injection.</li>
			<li>Open the NMR rotor by rotating clockwise the external screw mechanism which is coupled to the interior NMR cap bit.</li>
			<li>Allow the system to equilibrate to the desired gas pressures inside the NMR rotor and reseal the NMR rotor by rotating the external screw mechanism counterclockwise. A viewing window will assist in determining when the rotor is closed.</li>
			<li>Stop the liquid syringe pump injection and configure the valves to bypass the liquid injection line, disconnecting the pump from the system.</li>
			<li>Stop the flowing gas.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Purge the system with an inert gas to remove any potentially toxic or flammable gases.</li>
	<li>Stop any heating and allow the system to cool.</li>
	<li>Disconnect any heating tape and the thermocouple.</li>
	<li>Disconnect the inlet and outlet gas lines.</li>
	<li>Loosen the 6 bolts on the top of the exposure device to compromise the seal.</li>
	<li>Unclip the 2 clamping sections and remove them from the exposure device.</li>
	<li>Carefully lift the upper section up and off the lower section.</li>
	<li>Use an Allen wrench to loosen the rotor stage and draw it up with the threaded rod.</li>
	<li>Loosen the nut on the rotor stage and remove the rotor from the device component.</li>
	<li>Weigh the rotor to ensure the desired gas quantities are present.</li>
</ol>
4. Conducting the MAS NMR experiment
<ol>
	<li>Place the NMR rotor into the NMR coil on the NMR probe.</li>
	<li>Raise the probe into the magnet bore and lock it into place.</li>
	<li>Initiate sample spinning using the MAS control box and adjust to the desired rotor spinning rate.</li>
	<li>Use the computer to begin the tuning/match sequence on the desired channel.</li>
	<li>Adjust the tuning/match settings on the probe to optimize the probe electronics.</li>
	<li>Exit the tuning/match sequence on the computer and set up the desired experimental parameters (e.g., pulse sequence, experiment array, temperature, etc.).</li>
	<li>Collect the MAS NMR data.</li>
</ol>

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</ol>

The authors declare the following competing financial interests. J.Z.H and colleagues hold a patent on the rotor design (US9151813B2). J.Z.H., N.R.J., et al. have filed a provisional patent application on the exposure device.

The method of conducting MAS NMR spectroscopic measurements outlined herein represents the state of the art for conducting high-temperature, high-pressure MAS NMR. Such methods enable the observation of interactions occurring in vacuum atmospheres up to several hundred bar and from low temperatures (well below 0 &#176;C to 250 &#176;C) in a reliable, reproducible fashion. The ability to probe systems containing mixtures of solids, liquids, and gases under flexible chemical environments enables a wide range of experiments...

Spectroscopic analysis of samples is an analytical tool used to gain valuable information about materials of interest such as their chemical state, structure, or reactivity. In a simplistic view, nuclear magnetic resonance (NMR) is one such technique that utilizes a strong magnetic field to manipulate the spin state of atomic nuclei to better understand the chemical environment of the species of interest. The nuclear spin state refers to the relative direction of the magnetic moment induced by the motion of the spinning nucleus, a positively charged particle. In the absence of a magnetic field, the nuclear spins are randomly oriented but in the presence of a magnetic field, nuclear spins preferentially align with the external field of the magnet in a low energy spin state. This splitting of spin states to discrete energy values is known as the Zeeman effect. The difference between these energy levels (&#916;E) is modeled by Equation 1: 
<img alt="Equation 1" src="/files/ftp_upload/61794/61794equ01.jpg" /> 
where h is Plank&#8217;s constant, B0 is the strength of the external magnetic field and &#947; is the gyromagnetic ratio of the nucleus. The chemical environment of these spins also applies slight perturbations to these energy levels. Radio waves of corresponding frequencies can be used to excite the nuclei, which generates a transverse magnetization due to spins gaining phase coherence as longitudinal magnetization (based on the population of spins in parallel and anti-parallel states) is decreased. As the nuclei continue precessing about the axis of the magnetic field, the rotating magnetic movement creates a magnetic field that is also rotating and generating an electric field. This field modulates the electrons in the NMR detection coil, generating the NMR signal. Slight differences in the chemical environment of the nuclei in the sample affect the frequencies detected in the coil.
NMR analysis of solid samples introduces complexities not found in fluids. In fluids, the molecules tumble at fast rates, averaging the chemical environment spatially around the nuclei. In solid samples, no such averaging effect occurs, introducing an orientation-dependent chemical environment and broad spectral lines in the NMR signal. To mitigate these challenges, a technique known as magic angle spinning (MAS) is employed1,2. In MAS NMR, the samples are quickly rotated (several kilohertz) at an angle of 54.7356&#176; with respect to the external magnetic field using an external spinning mechanism to address the orientation-dependent (anisotropic) interactions of NMR. This substantially narrows the NMR features and enhances the spectral resolution by averaging the orientation-dependent terms of the chemical shift anisotropy, dipolar interactions, and quadrupolar interactions. Two notable exceptions do hinder the line narrowing abilities of MAS NMR. The first is strong homonuclear coupling sometimes present in 1H NMR that requires high spinning speeds (~70 kHz) to remove. However, the significantly elevated temperatures of the high temperature applications will greatly suppress the 1H homonuclear interaction by imparting enhanced thermal motion such that a much reduced sample spinning rate can be utilized for significantly enhanced spectral resolution. Furthermore, with the technology continuously evolving, rotors with smaller diameters can now be fabricated to achieve spinning rates far exceeding 5 kHz, which helps to further suppress the 1H homonuclear dipolar interactions. The second exception is residual second-order quadrupolar interactions for nuclei with spin that exceeds one-half since only the first order term is eliminated at the magic angle, leaving more complex lineshapes that can only be improved by stronger external magnetic fields. It should be emphasized that 2D MQMAS techniques can be readily incorporated into the current technology so that a true isotropic chemical shift spectrum can be obtained in a similar way as to the standard MQMAS experiments3.
MAS NMR has enabled detailed characterization of solid materials, strengthening the quality of observations. However, the necessity of spinning the samples in NMR rotors (the sample holder) at high rates also imposes challenges in conducting experiments at elevated temperatures and pressures which may be more relevant to the conditions of interest. It may, at times, be desirable to examine materials under conditions that are relatively harsh for NMR rotors. A number of efforts have successfully adapted liquid-state NMR technologies to conduct high-temperature, high-pressure NMR4,5,6,7; however, commercial rotor caps used for solid-state MAS NMR may be expelled from the rotor at high pressures, causing significant damage to the equipment. Such effects may be compounded by examining a decomposition reaction that greatly increases the pressure in the sample holder. As such, new designs are required to effectively and safely conduct in situ NMR experiments. For example, the rotor must adhere to several qualities for effective use in MAS NMR, namely non-magnetic, lightweight, durable, temperature resistant, low NMR background material, sealable, high-strength, and chemical resistant. The pressures the rotor must withstand are quite large. Not only must the rotor withstand the pressure of the sample contained within (e.g., high-pressure gas), the rotation of the device imparts centrifugal force which has its own contribution to the total system pressure8, PT, by equation 2: 
<img alt="Equation 2" src="/files/ftp_upload/61794/61794equ02.jpg" /> 
RI and RO are the inner and outer rotor radii, respectively, &#969; is the rotational frequency in radians per second, and Ps is the sample pressure.
A number of strategies have been developed to address these concerns9. Early examples resembled flame-sealed tubes10,11,12 or polymer inserts13,14, which were insufficient for extended, fine-controlled operation at elevated temperatures and pressures. Iterations to rotor designs have suffered from limitations in the maximum operating temperature imparted by the use of epoxy or sample volume reductions from ceramic inserts8,15,16. A recent technology reduces unit production costs by employing simple snap-in features in a commercial rotor sleeve, but offers relatively less control over the conditions with which it can operate17. The design employed herein is an all-zirconia, cavern-style rotor sleeve milled with a threaded top18. A cap is also threaded to allow for a secure seal. Reverse threading prevents sample rotation from loosening the zirconia cap and an O-ring constitutes the sealing surfaces. This rotor design is visible in Figure 1 and similar rotors and instructions to make them have been patented19. Such a strategy enables high mechanical strength, chemical resistance, and temperature tolerance.
These designs are suitable for temperatures and pressures of at least 250 &#176;C and 100 bar, limited in temperature by readily-available NMR probe technology. When combined with specialized sample preparation equipment, it represents a truly powerful technique that has been employed for far-reaching applications as carbon sequestration, catalysis, energy storage, and biomedicine20. Such equipment includes a way to pretreat the solid materials to remove unwanted surface species such as water. A furnace is often employed for this step. A dry box is typically used to load the solid samples into the NMR rotor. From there, the rotor is transferred into an exposure device which enables the rotor to be opened under a tightly controlled atmosphere to load a desired gas or mixture into the rotor. Such a device is depicted in Figure 2.

<table><tbody><tr><td>1) Preparation of Solids Samples</td><td></td><td></td><td></td></tr><tr><td>Gas maniforld</td><td></td><td></td><td></td></tr><tr><td>Gas Mass Flow Controllers</td><td></td><td></td><td></td></tr><tr><td>Vacuum Pump</td><td></td><td></td><td></td></tr><tr><td>Tube Furnace</td><td></td><td></td><td></td></tr><tr><td>Temperature Controller</td><td></td><td></td><td></td></tr><tr><td>Thermocouple</td><td></td><td></td><td></td></tr><tr><td>Quartz Tube</td><td></td><td></td><td></td></tr><tr><td>Isolation Valves</td><td></td><td></td><td></td></tr><tr><td>Quartz Wool</td><td></td><td></td><td></td></tr><tr><td></td><td></td><td></td><td></td></tr><tr><td>2) Loading solid samples into the rotor</td><td></td><td></td><td></td></tr><tr><td>Dry glove box</td><td></td><td></td><td></td></tr><tr><td>High-temperature, high-pressure NMR rotor</td><td></td><td></td><td></td></tr><tr><td>Sample funnel</td><td></td><td></td><td></td></tr><tr><td>Sample packing rod</td><td></td><td></td><td></td></tr><tr><td>Rotor holder</td><td></td><td></td><td></td></tr><tr><td>Analytical Balance</td><td></td><td></td><td></td></tr><tr><td>Microsyringe</td><td></td><td></td><td></td></tr><tr><td>Rotor cap bit</td><td></td><td></td><td></td></tr><tr><td></td><td></td><td></td><td></td></tr><tr><td>3) Addition of gases to the rotor</td><td></td><td></td><td></td></tr><tr><td>NMR loading chamber</td><td></td><td></td><td></td></tr><tr><td>Rotor stage and appropriately sized inserts</td><td></td><td></td><td></td></tr><tr><td>Vacuum Pump</td><td></td><td></td><td></td></tr><tr><td>Gas maniforld</td><td></td><td></td><td></td></tr><tr><td>Gas Mass Flow Controllers</td><td></td><td></td><td></td></tr><tr><td>Vacuum Pump</td><td></td><td></td><td></td></tr><tr><td>Heating Tape</td><td></td><td></td><td></td></tr><tr><td>Temperature Controller</td><td></td><td></td><td></td></tr><tr><td>Thermocouple</td><td></td><td></td><td></td></tr><tr><td>Allen wrench</td><td></td><td></td><td></td></tr><tr><td>Threaded rod</td><td></td><td></td><td></td></tr><tr><td>Wrenchs</td><td></td><td></td><td></td></tr><tr><td>Pressure Gauge</td><td></td><td></td><td></td></tr><tr><td>High-pressure syringe pump</td><td></td><td></td><td></td></tr><tr><td>Liquid syringe pump</td><td></td><td></td><td></td></tr><tr><td></td><td></td><td></td><td></td></tr><tr><td>4) Conducting the NMR experiments</td><td></td><td></td><td></td></tr><tr><td>MAS NMR probe</td><td></td><td></td><td></td></tr><tr><td>NMR spectrometer</td><td></td><td></td><td></td></tr><tr><td>Computer to control the spectrometer</td><td></td><td></td><td></td></tr></tbody></table>

high-temperature and high-pressure in situ magic angle spinning nuclear magnetic resonance spectroscopy

Nuclear magnetic resonance (NMR) spectroscopy represents an important technique to understand the structure and bonding environments of molecules. There exists a drive to characterize materials under conditions relevant to the chemical process of interest. To address this, in situ high-temperature, high-pressure MAS NMR methods have been developed to enable the observation of chemical interactions over a range of pressures (vacuum to several hundred bar) and temperatures (well below 0 &#176;C to 250 &#176;C). Further, the chemical identity of the samples can be comprised of solids, liquids, and gases or mixtures of the three. The method incorporates all-zirconia NMR rotors (sample holder for MAS NMR) which can be sealed using a threaded cap to compress an O-ring. This rotor exhibits great chemical resistance, temperature compatibility, low NMR background, and can withstand high pressures. These combined factors enable it to be utilized in a wide range of system combinations, which in turn permit its use in diverse fields as carbon sequestration, catalysis, material science, geochemistry, and biology. The flexibility of this technique makes it an attractive option for scientists from numerous disciplines.

The output from the NMR spectrometer takes the form of a free induction decay (FID) which is the time-domain signal from the excited spins as they relax back to thermodynamic equilibrium. Such an FID resembles Figure 3. When Fourier transformed from the time domain to the frequency domain (frequency to PPM by Equation 3, whereby the difference absolute frequency and a reference is divided by the carrier frequency of the NMR spectrometer), it represents the NMR spectrum for which each peak in...

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High-Temperature and High-Pressure In situ Magic Angle Spinning Nuclear Magnetic Resonance Spectroscopy

The molecular structures and dynamics of solids, liquids, gases, and mixtures are of critical interest to diverse scientific fields. High-temperature, high-pressure in situ MAS NMR enables detection of the chemical environment of constituents in mixed phase systems under tightly controlled chemical environments.

Nuclear magnetic resonance (NMR) spectroscopy represents an important technique to understand the structure and bonding environments of molecules. There ...

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Unit 1

Interpreting Nuclear Magnetic Resonance Spectra

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5.5K Views.  Pacific Northwest National Laboratory. De moleculaire structuren en dynamiek van vaste stoffen, vloeistoffen, gassen en mengsels zijn van cruciaal belang voor verschillende wetenschappelijke gebieden. Hoge temperatuur, hoge druk in situ MAS NMR maakt detectie van de chemische omgeving van bestanddelen in gemengde fase systemen onder strak gecontroleerde chemische omgevingen mogelijk.

Video: High-Temperature and High-Pressure In situ Magic Angle Spinning Nuclear Magnetic Resonance Spectroscopy

Atomic Scale Structural Studies of Macromolecular Assemblies by Solid-state Nuclear Magnetic Resonance Spectroscopy

Supramolecular protein assemblies play fundamental roles in biological processes ranging from host-pathogen interaction, viral infection to the propagation of neurodegenerative disorders. Such assemblies consist in multiple protein subunits organized in a non-covalent way to form large macromolecular objects that can execute a variety of cellular functions or cause detrimental consequences. Atomic insights into the assembly mechanisms and the functioning of those macromolecular assemblies remain often scarce since their inherent insolubility and non-crystallinity often drastically reduces the quality of the data obtained from most techniques used in structural biology, such as X-ray crystallography and solution Nuclear Magnetic Resonance (NMR). We here present magic-angle spinning solid-state NMR spectroscopy (SSNMR) as a powerful method to investigate structures of macromolecular assemblies at atomic resolution. SSNMR can reveal atomic details on the assembled complex without size and solubility limitations. The protocol presented here describes the essential steps from the production of 13C/15N isotope-labeled macromolecular protein assemblies to the acquisition of standard SSNMR spectra and their analysis and interpretation. As an example, we show the pipeline of a SSNMR structural analysis of a filamentous protein assembly.

Structures of supramolecular protein assemblies at atomic resolution are of high relevance because of their crucial roles in a variety of biological phenomena. Herein, we present a protocol to perform high-resolution structural studies on insoluble and non-crystalline macromolecular protein assemblies by magic-angle spinning solid-state nuclear magnetic resonance spectroscopy (MAS SSNMR).

Supramolecular protein assemblies play fundamental roles in biological processes ranging from host-pathogen interaction, viral infection to the ...

Biochemistry

High-Sensitivity Nuclear Magnetic Resonance at Giga-Pascal Pressures: A New Tool for Probing Electronic and Chemical Properties of Condensed Matter under Extreme Conditions

Nuclear Magnetic Resonance (NMR) is one of the most important techniques for the study of condensed matter systems, their chemical structure, and their electronic properties. The application of high pressure enables one to synthesize new materials, but the response of known materials to high pressure is a very useful tool for studying their electronic structure and developing theories. For example, high-pressure synthesis might be at the origin of life; and understanding the behavior of small molecules under extreme pressure will tell us more about fundamental processes in our universe. It is no wonder that there has always been great interest in having NMR available at high pressures. Unfortunately, the desired pressures are often well into the Giga-Pascal (GPa) range and require special anvil cell devices where only very small, secluded volumes are available. This has restricted the use of NMR almost entirely in the past, and only recently, a new approach to high-sensitivity GPa NMR, which has a resonating micro-coil inside the sample chamber, was put forward. This approach enables us to achieve high sensitivity with experiments that bring the power of NMR to Giga-Pascal pressure condensed matter research. First applications, the detection of a topological electronic transition in ordinary aluminum metal and the closing of the pseudo-gap in high-temperature superconductivity, show the power of such an approach. Meanwhile, the range of achievable pressures was increased tremendously with a new generation of anvil cells (up to 10.1 GPa), that fit standard-bore NMR magnets. This approach might become a new, important tool for the investigation of many condensed matter systems, in chemistry, geochemistry, and in physics, since we can now watch structural changes with the eyes of a very versatile probe.

Nuclear magnetic resonance is one of the most important spectroscopic tools. Here, the development of a new approach under high pressure, currently up to 10.1 GPa, is presented. This opens a new window into condensed matter physics and chemistry, where high-pressure research is of great importance.

Nuclear Magnetic Resonance (NMR) is one of the most important techniques for the study of condensed matter systems, their chemical structure, and their ...

Engineering

Dissolution Dynamic Nuclear Polarization Instrumentation for Real-time Enzymatic Reaction Rate Measurements by NMR

The main limitation of NMR-based investigations is low sensitivity. This prompts for long acquisition times, thus preventing real-time NMR measurements of metabolic transformations. Hyperpolarization via dissolution DNP circumvents part of the sensitivity issues thanks to the large out-of-equilibrium nuclear magnetization stemming from the electron-to-nucleus spin polarization transfer. The high NMR signal obtained can be used to monitor chemical reactions in real time. The downside of hyperpolarized NMR resides in the limited time window available for signal acquisition, which is usually on the order of the nuclear spin longitudinal relaxation time constant, T1, or, in favorable cases, on the order of the relaxation time constant associated with the singlet-state of coupled nuclei, TLLS. Cellular uptake of endogenous molecules and metabolic rates can provide essential information on tumor development and drug response. Numerous previous hyperpolarized NMR studies have demonstrated the relevancy of pyruvate as a metabolic substrate for monitoring enzymatic activity in vivo. This work provides a detailed description of the experimental setup and methods required for the study of enzymatic reactions, in particular the pyruvate-to-lactate conversion rate in presence of lactate dehydrogenase (LDH), by hyperpolarized NMR.

The sensitivity enhancement provided by dissolution dynamic nuclear polarization (DNP) enables following metabolic processes in real time by NMR and MRI. The characteristics and performances of a dedicated dissolution DNP setup designed for study enzymatic reactions are discussed.

The main limitation of NMR-based investigations is low sensitivity. This prompts for long acquisition times, thus preventing real-time NMR measurements of ...

Preparation of Fungal and Plant Materials for Structural Elucidation Using Dynamic Nuclear Polarization Solid-State NMR

This protocol shows how uniformly 13C, 15N-labeled fungal materials can be produced and how these soft materials should be proceeded for solid-state NMR and sensitivity-enhanced DNP experiments. The sample processing procedure of plant biomass is also detailed. This method allows the measurement of a series of 1D and 2D 13C-13C/15N correlations spectra, which enables high-resolution structural elucidation of complex biomaterials in their native state, with minimal perturbation. The isotope-labeling can be examined by quantifying the intensity in 1D spectra and the polarization transfer efficiency in 2D correlation spectra. The success of dynamic nuclear polarization (DNP) sample preparation can be evaluated by the sensitivity enhancement factor. Further experiments examining the structural aspects of the polysaccharides and proteins will lead to a model of the three-dimensional architecture. These methods can be modified and adapted to investigate a wide range of carbohydrate-rich materials, including the natural cell walls of plants, fungi, algae and bacteria, as well as synthesized or designed carbohydrate polymers and their complex with other molecules.

A protocol for preparing 13C,15N-labeled fungal and plant samples for multidimensional solid-state NMR spectroscopy and dynamic nuclear polarization (DNP) investigations is presented.

This protocol shows how uniformly 13C, 15N-labeled fungal materials can be produced and how these soft materials should be proceeded for solid-state NMR ...

Methods of Ex Situ and In Situ Investigations of Structural Transformations: The Case of Crystallization of Metallic Glasses

We demonstrate the use of two nuclear-based analytical methods that can follow the modifications of microstructural arrangement of iron-based metallic glasses (MGs). Despite their amorphous nature, the identification of hyperfine interactions unveils faint structural modifications. For this purpose, we have employed two techniques that utilize nuclear resonance among nuclear levels of a stable 57Fe isotope, namely M&#246;ssbauer spectrometry and nuclear forward scattering (NFS) of synchrotron radiation. The effects of heat treatment upon (Fe2.85Co1)77Mo8Cu1B14 MG are discussed using the results of ex situ and in situ experiments, respectively. As both methods are sensitive to hyperfine interactions, information on structural arrangement as well as on magnetic microstructure is readily available. M&#246;ssbauer spectrometry performed ex situ describes how the structural arrangement and magnetic microstructure appears at room temperature after the annealing under certain conditions (temperature, time), and thus this technique inspects steady states. On the other hand, NFS data are recorded in situ during dynamically changing temperature and NFS examines transient states. The use of both techniques provides complementary information. In general, they can be applied to any suitable system in which it is important to know its steady state but also transient states.

Methods of Ex Situ and In Situ Investigations of Structural Transformations: The Case of Crystallization of Metallic Glasses

Here, we present a protocol to describe ex situ and in situ investigations of structural transformations in metallic glasses. We employed nuclear-based analytical methods which inspect hyperfine interactions. We demonstrate the applicability of M&#246;ssbauer spectrometry and nuclear forward scattering of synchrotron radiation during temperature-driven experiments.

We demonstrate the use of two nuclear-based analytical methods that can follow the modifications of microstructural arrangement of iron-based metallic ...

High-Pressure NMR Experiments for Detecting Protein Low-Lying Conformational States

High-pressure is a well-known perturbation method that can be used to destabilize globular proteins and dissociate protein complexes in a reversible manner. Hydrostatic pressure drives thermodynamical equilibria toward the state(s) with the lower molar volume. Increasing pressure offers, therefore, the opportunities to finely tune the stability of globular proteins and the oligomerization equilibria of protein complexes. High-pressure NMR experiments allow a detailed characterization of the factors governing the stability of globular proteins, their folding mechanisms, and oligomerization mechanisms by combining the fine stability tuning ability of pressure perturbation and the site resolution offered by solution NMR spectroscopy. Here we present a protocol to probe the local folding stability of a protein via a set of 2D 1H-15N experiments recorded from 1 bar to 2.5 kbar. The steps required for the acquisition and analysis of such experiments are illustrated with data acquired on the RRM2 domain of hnRNPA1.

We provide a detailed description of the steps required to assemble a high-pressure cell, set up and record high-pressure NMR experiments, and finally analyze both peak intensity and chemical shift changes under pressure. These experiments can provide valuable insights into the folding pathways and structural stability of proteins.

High-pressure is a well-known perturbation method that can be used to destabilize globular proteins and dissociate protein complexes in a reversible ...

High-Resolution Studies of Proteins in Natural Membranes by Solid-State NMR

Membrane proteins are vital for cell function and thus represent important drug targets. Solid-state Nuclear Magnetic Resonance (ssNMR) spectroscopy offers a unique access to probe the structure and dynamics of such proteins in biological membranes of increasing complexity. Here, we present modern solid-state NMR spectroscopy as a tool to study structure and dynamics of proteins in natural lipid membranes and at atomic scale. Such spectroscopic studies profit from the use of high-sensitivity ssNMR methods, i.e., proton-(1H)-detected ssNMR and DNP (Dynamic Nuclear Polarization) supported ssNMR. Using bacterial outer membrane beta-barrel protein BamA and the ion channel KcsA, we present methods to prepare isotope-labeled membrane proteins and to derive structural and motional information by ssNMR.

This work details robust basic routines on how to prepare isotope-labeled membrane protein samples and analyze them at high-resolution with modern solid-state NMR spectroscopy methods.

Membrane proteins are vital for cell function and thus represent important drug targets. Solid-state Nuclear Magnetic Resonance (ssNMR) spectroscopy ...

Protocol for NMR 15N Relaxation and hetNOE Experiments in Protein Dynamics Study

NMR 15N Relaxation Experiments for the Investigation of Picosecond to Nanoseconds Structural Dynamics of Proteins

Nuclear magnetic resonance (NMR) spectroscopy allows studying proteins in solution and under physiological temperatures. Frequently, either the amide groups of the protein backbone or the methyl groups in side chains are used as reporters of structural dynamics in proteins. A structural dynamics study of the protein backbone of globular proteins on 15N labeled and fully protonated samples usually works well for proteins with a molecular weight of up to 50 kDa. When side chain deuteration in combination with transverse relaxation optimized spectroscopy (TROSY) is applied, this limit can be extended up to 200 kDa for globular proteins and up to 1 MDa when the focus is on the side chains. When intrinsically disordered proteins (IDPs) or proteins with intrinsically disordered regions (IDRs) are investigated, these weight limitations do not apply but can go well beyond. The reason is that IDPs or IDRs, characterized by high internal flexibility, are frequently dynamically decoupled. Various NMR methods offer atomic-resolution insights into structural protein dynamics across a wide range of time scales, from picoseconds up to hours. Standard 15N relaxation measurements overview a protein&#39;s internal flexibility and characterize the protein backbone dynamics experienced on the fast pico- to nanosecond timescale. This article presents a hands-on protocol for setting up and recording NMR 15N R1, R2, and heteronuclear Overhauser effect (hetNOE) experiments. We show exemplary data and explain how to interpret them simply qualitatively before any more sophisticated analysis.

Author Spotlight: Exploring Intrinsically Disordered Protein Dynamics Through NMR Relaxation Experiments

Nuclear magnetic resonance (NMR) spectroscopy can characterize structural protein dynamics in a residue-specific manner. We provide a hands-on protocol for recording NMR 15N R1 and R2 relaxation and {1H}-15N heteronuclear Overhauser effect (hetNOE) experiments, sensitive to the picoseconds to nanoseconds timescale.

Nuclear magnetic resonance (NMR) spectroscopy allows studying proteins in solution and under physiological temperatures. Frequently, either the amide ...

Cryogenic Sample Loading into a Magic Angle Spinning Nuclear Magnetic Resonance Spectrometer that Preserves Cellular Viability

Dynamic nuclear polarization (DNP) can dramatically increase the sensitivity of magic angle spinning (MAS) nuclear magnetic resonance (NMR) spectroscopy. These sensitivity gains increase as temperatures decrease and are large enough to enable the study of molecules at very low concentrations at the operating temperatures (~100 K) of most commercial DNP-equipped NMR spectrometers. This leads to the possibility of in-cell structural biology on cryopreserved cells for macromolecules at their endogenous levels in their native environments. However, the freezing rates required for cellular cryopreservation are exceeded during typical sample handling for DNP MAS NMR and this results in loss of cellular integrity and viability. This article describes a detailed protocol for the preparation and cryogenic transfer of a frozen sample of mammalian cells into a MAS NMR spectrometer.

Presented here is a protocol for cryogenic transfer of frozen samples into the dynamic nuclear polarization (DNP) magic angle spinning (MAS) nuclear magnetic resonance (NMR) probe. The protocol includes directions for rotor storage prior to the experiment and directions for viability measurements before and after the experiment.

Dynamic nuclear polarization (DNP) can dramatically increase the sensitivity of magic angle spinning (MAS) nuclear magnetic resonance (NMR) spectroscopy. ...

Biology

Nuclear Magnetic Resonance (NMR) Spectroscopy

Source: Laboratory of Dr. Henrik Sund&#233;n &#8211; Chalmers University of Technology

Nuclear magnetic resonance (NMR) spectroscopy is a vital analysis technique for organic chemists. With the help of NMR, the work in the organic lab has been facilitated tremendously. Not only can it provide information about the structure of a molecule but also determine the content and purity of a sample. Compared with other commonly encountered techniques for organic chemists &#8212; such as thermal analysis and mass spectrometry (MS) &#8212; NMR is a non-destructive method that is valuable when recovery of the sample is important.
One of the most frequently used NMR techniques for an organic chemist is proton (1H) NMR. The protons present in a molecule will behave differently depending on its surrounding chemical environment, making it possible to elucidate its structure. Moreover, it is possible to monitor the completion of a reaction by comparing NMR spectra of the starting material to that of the final product.
This video exemplifies how NMR spectroscopy can be used in the everyday work of an organic chemist. The following will be shown: i) preparation of an NMR sample. ii) Using 1H NMR to monitor a reaction. iii) Identifying the product obtained from a reaction with 1H NMR. The reaction that will be shown is the synthesis of an E-chalcone (3) from an aldehyde (1) and a ketone (2) (Scheme 1).1
<img alt="Scheme 1" src="/files/ftp_upload/5680/5680scheme1.jpg" /> 
Scheme 1. Synthesis of (2E)-3-(4-methoxyphenyl)-1-(4-methylphenyl)-2-propen-1-one.

NMR Spectroscopy: Principle, NMR Active Nuclei, Applications

Source: Laboratory of Dr. Henrik Sund&#233;n &#8211; Chalmers University of Technology

Nuclear magnetic resonance (NMR) spectroscopy is a vital analysis ...

High-Temperature and High-Pressure In situ Magic Angle Spinning Nuclear Magnetic Resonance Spectroscopy

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